Development of an Optical Fiber Biosensor with Nanoscale Self-Assembled Affinity Layer

217 Pages·2014·12.3 MB·English

Development of an Optical Fiber Biosensor
with Nanoscale Self-Assembled Affinity Layer

Ziwei Zuo

Dissertation submitted to the Faculty of the
Virginia Polytechnic Institute and State University
In partial fulfillment of the requirements for the degree of

Doctor of Philosophy
In
Physics

James R. Heflin, Chairman
Jean J. Heremans
Hans D. Robinson
Thomas J. Inzana

Nov 20, 2013
Blacksburg, Virginia

Keywords: long-period-gratings (LPG), ionic self-assembled multilayer (ISAM) films, fiber optical sensor,
methicillin-resistant S. aureus (MRSA), Brucellosis, Francisella tularensis, monoclonal antibody (Mab)

Copyright © 2013 Ziwei Zuo
Abstract
Development of an Optical Fiber Biosensor with
Nanoscale Self-Assembled Affinity Layer

Ziwei Zuo

Optical sensor systems that integrate Long-Period-Gratings (LPG) as the detection arm have
been proven to be highly sensitive and reliable in many applications. With increasing public
recognition of threats from bacteria-induced diseases and their potential outbreak among densely
populated communities, an intrinsic, low-cost biosensor device that can perform quick and
precise identification of the infection type is in high demand to respond to such challenging
situations and control the damage those diseases could possibly cause.
This dissertation describes the development of a biosensor platform that utilizes polymer
thin films, known as ionic self-assembled multilayer (ISAM) films, to be the sensitivity-
enhancing medium between an LPG fiber and specific, recognition layer. With the aid of cross-
linking reactions, monoclonal antibodies (IgG) or DNA probes are immobilized onto the surface
of the ISAM-coated fiber, which form the core component of the biosensor.
By immersing such biosensor fiber into a sample suspension, the immobilized antibody
molecules will bind the specific antigen and capture the target cells or cell fragments onto the
surface of the fiber sensor, resulting in increasing the average thickness of the fiber cladding and
changing the refractive index of the cladding. This change occurring at the surface of the fiber
results in a decrease of optical power emerging from the LPG section of the fiber. By comparing
the transmitted optical power before and after applying the sample suspension, we are able to
determine whether or not certain bacterial species have attached to the surface of the fiber, and as
a consequence, we are able to determine whether or not the solution contains the targeted
bacteria.
This platform has the potential for detection of a wide range of bacteria types. In our study,
we have primarily investigated the sensitivity and specificity of the biosensor to methicillin-
resistant Staphlococcus aureus (MRSA). The data we obtained have shown a sensitive threshold
at as low as 102 cfu/ml with pure culture samples. A typical MRSA antibody-based biosensor
assay with MRSA sample at this concentration has shown optical power reduction of 21.78%. In
a detailed study involving twenty-six bacterial strains possessing the PBP2a protein that enables
antibiotic resistance and sixteen strains that do not, the biosensor system was able to correctly
identify every sample in pure culture samples at concentration of 104 cfu/ml. Further studies
have also been conducted on infected mouse tissues and clinical swab samples from human ears,
noses, and skin, and in each case, the system was in full agreement with the results of standard
culture tests. However, the system is not yet able to correctly distinguish MRSA and non-MRSA
infections in clinical swab samples taken from infected patient wounds. It is proposed that
nonspecific binding due to insufficient blocking methods is the key issue.
Other bacterial strains, such as Brucella and Francisella tularensis have also been studied
using a similar biosensor platform with DNA probes and antibodies, respectively, and the
outcomes are also promising. The Brucella DNA biosensor is able to reflect the existence of 3
Brucella strains at 100 cfu/ml with an average of 12.2% signal reduction, while negative control
samples at 106cfu/ml generate an average signal reduction of -2.1%. Similarly, the F. tularensis
antibodies biosensor has shown a 25.6% signal reduction to LVS strain samples at 100 cfu/ml,
while for negative control samples at the same concentration, it only produces a signal reduction
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of 0.05%. In general, this biosensor platform has demonstrated the potential of detecting a wide
range of bacteria in a rapid and relatively inexpensive manner.

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Acknowledgement
First of all, I would like to thank my advisor, Dr. James R. Heflin, for giving me the
opportunity to work on this project, and for his precious research advice, enormous support and
encouragement through years of laboratory work. Without his insight and expertise on the
obstacles I have encountered in this project, I could never be able to finish my research on this
project. I can’t express enough appreciation for all the time Dr. Heflin has devoted in my
research, and I would like to dedicate this dissertation to him.
I also thank Dr. Thomas J. Inzana for his valuable advice and suggestions on the
biochemistry issues we met during the research, and without his support and Dr. Aloka B.
Bandara’s hard work on providing the strain samples, this project won’t be possible. I value
highly of our collaboration, and I would like to give my deep thanks to them.
Thanks to my committee members: Dr. Hans Robinson and Dr. Jean J. Heremans, for
offering all the help I asked for. Thanks to Dr. Jimmy Ritter, for his support and assistance in this
research project and offering great ideas on issues we have constantly come across.
And I can’t afford to forget to give thanks to Mr. Moataz Khalifa, for offering me the
opportunity to conduct AFM studies, and accompany me over many unforgettable nights in the
laboratory. Without his expertise on AFM, his impressive smell and humor, I would enjoy much
less of my laboratory time. Also thanks to Ms. Kelly Mccutcheon for helping me oversee the
experiments when I have to focus on the dissertation writings.
During my years in pursuing my degrees in Virginia Tech, I have met so many great faculty
and staff who I want to thank. I want to give my special thanks to Ms. Christa Thomas, for her
great help during all these years, on so many trivial or important matters. Thanks to Dr. Uwe
v
Täuber for his wonderful advice during my first year in the graduate program of Physics
department.
I would like to thank my parents, Mr. Ming Zuo and Ms. Hui Cheng. Their unending
support and love have given me great encouragement to carry on my study in Virginia Tech. And
I thank them for offering me good education and opportunities without having any regrets or
asking for returns.
I am deeply thankful to have received many helps and spiritual nourishment from Dr.
Johnny Yu and Dr. Jessie Yu-Chen, they have made my stay in Blacksburg as like home. Also
thanks to Dr. Y.A Liu, Dr. Chao Shang, Dr. Liwu Li, Dr. Peter Lo, Dr. Caisy Ho, Dr. Paul
Carlier, Dr. Don Mckeon, Dr. Randall Cliff. You have been great example to me and have taught
me lessons I won’t get anywhere else.
And thanks to my good old friends, Dr. Qiang Li, Mr. Lester Jay, Dr. Jay Sullivan, Dr.
Tongli Zhang, Mr. Lifeng Li, Mr. Gang Wu, Ms. Luman Chen, Ms. Yanjun Ma, Ms. Ying Xu,
Mr. Di Zeng, Mr. Yao Li, Mr. Renyuan Fu, Mr. Waifong Chan, Ms. Yiming Tang, Dr. Chih-ling
Liou, Ms. Yueying Yu, Mr. Yitao Zhu, Ms. Yinglian Yang, Ms. Bing Yan, Mr. Christoph Paulus,
Dr. Ni Shen, Dr. Cheng Ma, Mr. Zhipeng Tian, and many more that I can’t list here. Friendship
never will grow old with you fellows. And special thanks to Ms. Qing Li and Dr. Jing Zhang.
Thanks to my best “bad” friend Mr. Zhao Liu and Mr. Xiao Lv, killing time with you can’t be
more fun. And thanks to Dr. Danielle Jao for giving my great advice on graduation.
And I can’t forget my dear friend Mr. Chuck Schumann, I know you are happy for me in
heaven.

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Contents
Chapter 1 Introduction .......................................................................... 1

1.1 Review of Biosensing Technology ..................................................................................................... 1

1.2 Current Development of Optical Biosensors ...................................................................................... 5

1.3 Turnaround Point Long-Period-Gratings with Ionic Self-Assembled Multilayers ............................ 8

1.4 Outline .............................................................................................................................................. 13

Chapter 2 Long-Period-Gratings (LPG) based Biosensor .......................... 15

2.1 Long Period Gratings ....................................................................................................................... 15

2.1.1 Single-mode Fiber ...................................................................................................................... 15

2.1.2 Cladding Mode ........................................................................................................................... 17

2.1.3 Mode Coupling and Phase-matching in a Single Mode Fiber ................................................... 18

2.1.4 Phase Matching Curve ............................................................................................................... 21

2.1.5 Turnaround Point in an LPG Fiber ............................................................................................ 27

2.1.6 Applications of the TAP LPG as a Sensor ................................................................................. 39

2.2 Ionic Self-Assembled Multilayers (ISAM) ...................................................................................... 43

2.2.1 Introduction to Ionic Self-assembled Multilayer Thin Films .................................................... 43

2.2.2 Structure and Deposition of ISAM ............................................................................................ 45

2.2.3 PAH and PCBS Polyelectrolytes ............................................................................................... 50

2.2.4 AFM Studies of the ISAM Thin Film ........................................................................................ 55

2.2.5 Discussion of the ISAM Thin Film Coating Technique ............................................................ 57

2.3 Cross-linking Chemistry .................................................................................................................. 58

2.3.1 Immobilization of the Biological Molecules on the ISAM Thin Film ...................................... 58

2.3.2 Cross-linking Conditions and Efficiency ................................................................................... 64

2.3.3 Common Cross-linking Reactions ............................................................................................. 67

2.3.4 Application of Carboxyl-to-amine Cross-linking Reaction in Building the TAP-LPG Fiber
Biosensor ............................................................................................................................................. 79

2.4 Implemention of immunology Techniques for Biosensing .............................................................. 81

2.4.1 Overview of Immunology ......................................................................................................... 81

2.4.2 Biological Receptor .................................................................................................................. 85

2.4.3 Immunoassays and Immunological Tests ................................................................................. 87

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Chapter 3 Experimental Detail of Optical Fiber Sensor for Detecting and
Differentiating various Bacteria ........................................................... 95

3.1 Immunoassay Based Fiber Biosensor .............................................................................................. 95

3.2 The Components of the Fiber Biosensor System ............................................................................. 97

3.2.1 Biosensor System Setup ............................................................................................................. 97

3.2.2 White Light Source .................................................................................................................. 100

3.2.3 Optical Spectrum Analyzer ...................................................................................................... 103

3.2.4 Stability and Repeatability Study of the Biosensor System ..................................................... 103

3.2.5 Optical Fiber Preparation ......................................................................................................... 105

3.2.6 Test of LPG Sensor .................................................................................................................. 108

Chapter 4 Application of the LPG Biosensor for MRSA Detection .......... 112

4.1 Introduction to Methicillin-resistant Staphylococcus aureus (MRSA) .......................................... 112

4.2 MRSA biosensor using monoclonal antibodies as receptor ........................................................... 114

4.2.1 Monoclonal Antibodies to MRSA .......................................................................................... 114

4.2.2 Experimental Methods and Procedure .................................................................................... 116

4.2.3 Preparation of the Sample Suspensions .................................................................................. 124

4.2.4 Biosensor Assay Results with Pure Culture Samples ............................................................. 126

4.2.5 Biosensor Assay Results with Human Subject Samples ......................................................... 136

4.2.6 Biosensor Assay Results with Mice Tissue Samples .............................................................. 138

4.2.7 Biosensor Assay Results with Clinical Swab Samples ........................................................... 142

4.2.8 Statistical Importance of the Biosensor Assays ...................................................................... 144

4.3 The MRSA Biosensor Using DNA Probe as Receptor .................................................................. 149

4.3.1 Replacing Mab with DNA-probes for MRSA Biosensor ....................................................... 149

4.3.2 DNA Probe Biosensor Assembly and Testing Methods ......................................................... 151

4.3.3 DNA Biosensor Assay Results with Pure Culture Samples ................................................... 157

4.3.4 DNA Probe Biosensor Assay Results with Clinical Swab Samples ....................................... 160

4.4 AFM Imaging Studies .................................................................................................................... 161

Chapter 5 Identification of Other Bacteria with the LPG Biosensor ......... 172

5.1 Adaptive Biosensor Platform ......................................................................................................... 172

5.2 The F. tularensis Biosensor ........................................................................................................... 172

5.3 The Brucella Biosensor .................................................................................................................. 175

5.4 The H. somni Biosensor .................................................................................................................. 179

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Chapter 6 Conclusion and Future Work ................................................ 184

6.1 Conclusion ....................................................................................................................................... 184

6.2 Future Work .................................................................................................................................... 189

6.2.1 Introducing Protein-G to Improve Bioconjugation of Antibodies ........................................... 190

6.2.2 Improvement of the DNA Biosensor with Swab Tests ............................................................ 191

6.2.3 Statistical Significance and Stability Studies ........................................................................... 192

6.2.4 Regenerating the Biosensor ..................................................................................................... 192

6.2.5 Determination of the Necessary Assay Length ........................................................................ 193

Bibliography .................................................................................... 194

List of Figures

Figure 2-1: a) A typical structure of a step-index optical fiber: (A) core of the fiber, (B)
cladding region, and (C) the buffer jacket protecting the fiber. b) Cross sectional view of
a step-index single-mode fiber (SMF). ................................................................................. 15

Figure 2-2 : a) Multimode fiber is able to support multiple modes within its core, and these
modes will spread out and lose its shape, and will be received separately at the receiving
point. b) Single-mode fiber. Source:[44]. ............................................................................. 17

Figure 2-3: a) a periodic perturbation grating in the core of the fiber, with the grating period
Λ, with the original core refractive index as n2 and the grating region refractive index n3.
The cladding refractive index is n and the surrounding medium’s refractive index isn . b)
1 0
a rectangular profile of the refractive index change in the core, where along every Λ in
length, the core refractive index n will step-jump to n and back to n , periodically.
2 3 2
Source: [48]. ......................................................................................................................... 19

Figure 2-4: Power conversion rateR(z). a) at z = 1.47, and δ/η = 0, 99% of the core mode
power at the resonant wavelength has been transferred to the cladding mode. While at z
= 0.8, at most only 50% of the core mode power is lost. b) the power conversion along
the propagation direction (z-axis). ........................................................................................ 23

Figure 2-5: Phase matching curves of the simulated LPG fiber. Each curve, from top to
bottom, refers to the phase matching conditions between the fundamental LP mode and
01
the coupled cladding LP mode (m from 2 to 12 in this figure). Source:[58]. ................... 25

0m
ix
Figure 2-6: Transmission spectrum of a typical LPG. Multiple dips can be observed in this
spectrum, indicating the resonant coupling between the core mode and four separate
cladding modes within the range of 1100 nm to 1600 nm. Source: [59]. ............................ 26

Figure 2-7: Coupling to different modes can deliver distinct behavior on the increment of the
cladding radius. The horizon line indicates the grating period in the simulated LPG fiber,
d refers to the additional thickness added to the cladding. n =1.8 is the refractive index
3
of the material added to the cladding. The PMC shifts upwards with thicker materials
over the cladding. a) Monotonic PMC of the LP coupling mode results in a resonant
0,4
wavelength shifting in the transmission spectrum. b) A TPA PMC of the LP coupling
0,12
mode delivers a power loss peak shifting along a single wavelength at 1550 nm.
Source:[58]. .......................................................................................................................... 28

Figure 2-8: PMC location to the grating periods reveals the corresponding transmission
spectrum. a) Grating periods with various distances to the TAP of the LPG. Horizontal
line indicates the grating periods, from bottom to top are 145.3µm, 146.3µm, 147.3µm,
147.4µm, 147.5µm and 147.8µm, respectively. b) The corresponding transmission
spectrum based on the PMC. Source:[58]. ........................................................................... 32

Figure 2-9: (A) a TAP LPG transmitted power loss evolution with more adsorbed materials
deposited on the cladding of the fiber. (B) Phase I, TAP and phase II are displayed under
the PMC view. The fiber is tuned slightly before hitting TAP. In phase I, with a few
nanometers ambient materials added to the cladding, the transmitted power loss
increases exponentially. Then at one point between 8 nm and 12 nm, the phase-
matching-condition is met, and then in phase II, detuning takes place and transmitted
power loss soon recovered. ................................................................................................... 34

Figure 2-10: Curve trajectory fitting and comparison in Phase I and Phase II. a) in Phase-I
before hitting the TAP, the transmitted light losses power exponentially when the
thickness of cladding rises. b) in Phase-II, after hitting the TAP, the detuning occurs, and
the mode-coupling vanishes as the power losses recovers from the dip. ............................. 35

Figure 2-11: The insensitive region at the TAP. Thickness of each ambient layers are
considered constant. Through layer 1 to layer 3, the fiber stays in phase I region and the
transmitted light loses power. Similarly, through layer 4 to layer 5, the fiber stays in
phase II region, and the light regains. However, between layer 3 and layer 4, the fiber is
right at the TAP, and the change of the power is slow given same amount of material is
added. (From Eric Carlson and Dr. Ritter’s study) .............................................................. 38

Figure 2-12: LPG temperature sensor developed by V. Bhatia (From [16]), and X. Shu (From
[17]) a) Bhatia’s sensor showed the transmission spectra readings of the single
attenuation peak locations under 22.7, 49.1, 74.0, 100.9, 127.3 and 149.7 ◦C respectively.
The linear shifting of the wavelength at different bands. Bands A-D are located at 1608.6
nm, 1332.9 nm, 1219.7 nm and 1159.6 nm respectively at 31.2 °C. b) Shu’s sensor
x